The phosphoenolpyruvate:sugar phosphotransferase system (PTS) is a complex enzyme system which in Salmonella typhimurium and Escherichia coli catalyzes the concomitant transmembrane transport and phosphorylation of its sugar substrates. It also regulates the activities of several independently functioning carbohydrate permeases, including the lactose permease of E. coli. Recent studies in our laboratory have provided new information regarding the mechanisms by which the PTS functions in the catalysis and regulation of sugar uptake. The purpose of the proposed research is to test our current hypotheses and to establish the correct mechanisms. Both biochemical and genetic approaches will be taken. A. PTS-mediated sugar transport. We plan to: 1) study the kinetics of the newly discovered Enzyme II catalyzed transphosphorylation reaction in vitro; 2) attempt functional dissection of the Enzyme II by genetic and biochemical means; 3) characterize PTS-catalyzed exchange transport and group translocation in bacterial membrane vesicles; 4) purify an Enzyme II and reconstitute transphosphorylation activity in isolated liposomes. B. PTS-mediated regulation of carbohydrate transport. We plan to: 1) isolate and characterize the lactose permease protein from wild type and lac-specific transport regulation resistant mutants; 2) characterize crrA and crrB mutations genetically and biochemically, and determine if other genetic regulatory loci adjacent to the pts operon exist; 3) study the newly discovered phenomenon of PTS-mediated transport regulation desensitization; 4) attempt to utilize membrane vesicles to identify the proteins directly responsible for PTS-mediated transport regulation.